PROJECT SUMMARY Type II diabetics are at very high risk for cardiovascular diseases, and ~30% of diabetics over age 50 suffer from peripheral artery disease (PAD). PAD affects ~10 million Americans and has a devastating clinical impact; current treatments often fail to provide long-term benefits, and amputation is the final option for patients with critical limb ischemia. More than 90% of non-trauma related limb amputations in the US are due to PAD. Arteriogenesis, the ability of the vasculature to bypass occlusion via enlargement of pre-existing collateral arteries, could improve blood flow in PAD patients; unfortunately, type II diabetes inhibits arteriogenesis. Since stimulation of arteriogenesis would be of enormous value in diabetics with PAD, it is essential that we comprehend 1) key mechanisms of arteriogenesis and 2) how these are blocked by type II diabetes. Placental growth factor (PLGF) specifically induces arteriogenesis and is a key initiating factor in the process. However, little is known about how PLGF levels are controlled. We made several key discoveries under the prior award that revealed a novel mechanism linking upregulation of PLGF to the arteriogenic signal of fluid shear stress (FSS). Furthermore, we established that a Western diet inhibits occlusion-induced PLGF expression in skeletal muscle, and gained insights into the mechanism involved. The overall objectives of this application are 1) to further pursue our knowledge of mechanisms regulating PLGF, and 2) to reveal the mechanism by which a Western diet impairs PLGF regulation. Our central hypotheses are: 1) FSS upregulates PLGF via a novel iron-dependent pathway; and 2) a Western diet inhibits PLGF expression by inducing sustained hyperglycemia. These hypotheses are strongly supported by our published results and preliminary data. We will accomplish three Specific Aims. Specific Aim 1 will expand our understanding of the molecular mechanism for upregulation of PLGF by fluid shear stress in vitro. These studies utilize an endothelial cell/smooth muscle cell co-culture model; pulsatile FSS is applied with a cone and plate device. This model was established in our lab under the prior award. Specific Aim 2 will extend our knowledge of the molecular mechanism for upregulation of PLGF by FSS from in vitro to ex vivo and in vivo systems. Models for these studies are isolated mouse peripheral arterioles and gradual femoral artery occlusion in mice; both are established in our lab. Specific Aim 3 will discover molecular mechanism(s) by which a Western diet causes dysfunctional PLGF regulation. These studies are done in cultured cells, and in isolated peripheral arterioles and ischemic hindlimbs of mice exhibiting a range of metabolic dysfunction induced by long-term Western diet consumption. These studies will reveal novel mechanisms regulating expression of this key arteriogenic growth factor and will identify new targets for potential therapeutic interventions to enhance arteriogenesis in diabetic PAD.